The horseshoe crabs Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas and Carcinoscorpius rotundicauda are phylogenetically primitive marine arthropods which have not evolved significantly over the past 300 million years (1).
The horseshoe crab has an open circulatory system containing blue haemolymph, and the only formed element present in the haemolymph is a cell called the amoebocyte (2).
The use of the Limulus amoebocyte lysate (LAL) as an in vitro test for endotoxin resulted directly from the important observation made by Bang (3). He observed that the horseshoe crab underwent a type of disseminated intravascular coagulation (DIC) when generalized infection occurred with marine gram-negative bacteria. This initial in vivo observation was later extended by the discovery that clotting of Limulus hemolymph could be produced in vitro by addition of either viable gram-negative bacteria or purified endotoxin from the cell wall of gram-negative bacteria (2). It was also discovered by the same researchers (1) that the amoebocyte was the source of all of the factors necessary for hemolymph coagulation.
The current application of the test as an in vitro assay for endotoxin is based on the fact that physical disruption of amoebocytes which have been isolated from the haemolymph by centrifugation yields a suspension (LAL) containing the coagulation components which may then only be activated by bacterial endotoxin. Application of this principle has made the LAL test the most sensitive method available for the detection of gram-negative bacteria, endotoxin and lipopolysaccharide (LPS). LAL prepared by current purification methods can reliably detect 0.1 ng/ml of purified Escherichia coli standard endotoxin. LAL has been shown to detect either bound (cell-associated) or free endotoxin (4) incorporated in the cell walls of virtually all gram-negative bacteria. However, endotoxin prepared by different extraction procedures, from different species, may vary widely in reactivity (5,6). For these reasons, a reference standard endotoxin (RSE) has been prepared by the U.S. Food and Drug Administration (USFDA) as a means for standarization of LAL and rabbit pyrogenicity tests (Unites States Pharmacopoeia XX) (7).
Amoebocyte lysate of similar reactivity to endotoxin can be prepared from all 4 species of horseshoe crabs.
The clotting processes in Limulidae
The currently known clotting processes in Limulidae are summarized in Table 1.
TABLE 1 ______________________________________ CLOTTING PROCESS IN LIMULIDAE ______________________________________ ##STR1## ##STR2## ##STR3## ______________________________________
Cellular coagulogen: Coagulogen consists of a polypeptide chain with internal disulfide bonds which are important for the stability of the polymerizable form of the molecule (8). In Limulus polyphemus, coagulogen was found to consist of 220 amino acids with half-cystine content of 18 residues (9). No free SH group was detected; glycine appears to be the only N-terminal residue, and serine its C-terminal residue (9). Coagulogen is always converted by a serine protease enzyme. After clotting, the gel protein displays a helical structure in electron microscopy (10). In Tachypleus tridentatus, the coagulogen comprises 132-135 amino acids including high levels of basic amino acids, with N-terminal alanine and C-terminal phenylalaine. In Limulus, clot formation seems to involve the cleavage of the single Arg-Lys peptide bond on the coagulogen (11,9). The N-peptides interact among themselves in a non-covalent fashion to form the insoluble clot. In Tachypleus tridentatus, the enzymatic formation of gel involves limited proteolysis of the Arg-Gly and Arg-Thr peptide linkages located in the N-terminal portion of coagulogen, thus releasing peptide (12,13). The C-fragment of Limulus mainly contains glutamic and aspartic acids (14). Whereas Liu et al. (9) detected a C-peptide with 45 amino acids in this species, Nakamura et al. (13) and Shishikura et al. (15) claimed that this C-peptide had 28 amino acid residues arranged in a species-specific sequence.
Clotting enzyme: The clotting enzyme is a serine protease enzyme. It exists in two active forms with molecular weights of 78,000 and 40,000, respectively, and a very similar amino acid composition, indicating a monomer-dimer relationship (9). In Tachypleus tridentatus, the unreduced clotting enzyme is described as a glycoprotein with a molecular weight of 42,000 which aggregates to form a protein with a MW of 350,000. The clotting enzyme originates from an inactive pro-clotting enzyme. Pro-clotting enzyme can be active via two independent pathways (Table 1): by the LPS of gram-negative bacteria, or by the (1-3)-.beta.-D-glucans from the cell walls of certain fungi and algae.
LPS-mediated coagulation: Firstly, it was demonstrated that endotoxin or LPS activates a pro-clotting enzyme of the serine protease type (16,11). Secondly, an additional Factor B or pro-activator was also found to be involved in the LPS-induced coagulation of LAL since it activated the pro-clotting enzyme (17,18). This activation probably involves limited proteolysis, i.e. of an arginyl or lysyl-X bond of the pro-clotting enzyme (18). Finally, this pro-activator was observed to be converted to an active Factor B or activator (i.e. trypsin-type serine protease) by another proteolytic enzyme called protease N (Factor C in T. tridentatus) which seems to be LPS-dependent (18,19). These successive findings indicate that this coagulation process represent a complex enzyme cascade which might also include other unknown factors (Table 1).
Anti-clotting factors: An 80 Kd protein form the amoebocyte membrane specifically binds the endotozin or LPS (9,20). According to the authors, this receptor protein can recognize and immobilize small quantities of LPS in Limulus blood without massive intravascular coagulation. In addition, an anticoagulant (anti-LPS factor) which inhibits LPS-inducted coagulation is present in the amoebocytes from the hemolymph of Tachypleus tridentatus and Limulus polyphemus (21). This anticoagulant inhibits the activation of Factor B, but not its activity. The other mediated coagulation pathway is not affected by the anti-LPS factor (21).
(1-3).beta.-D-glucan-mediated coagulation: Both the antitumor agent -(1-3)-.beta.-D-glucan and other antitumor polysaccharies have a potent ability to cause gelatin of LAL (22) by activating a factor G which acts on pro-clotting enzyme (23).